This invention relates to analytical methods for determining the presence of an analyte at a predetermined concentration in a test sample. More particularly, the invention concerns such a determination wherein the presence of the analyte at or above the concentration of interest is indicated by the appearance of a preselected spectrophotometric response, such as the appearance of color. Such determination is accordingly self-indicating because comparators or standards are unnecessary. In a further embodiment, the invention concerns visually read assays requiring optimization of color resolution of the indicator response over the range of analyte concentrations of significance.
Test methods are well known for determining the concentration of an analyte in a liquid test sample based on a spectrophotometric response produced by chemical reaction between the analyte of interest and an appropriate reagent/indicator system. The spectrophotometric response is usually a color change that is measured instrumentally or visually observed. Conventional tests provide quantitation of the amount or concentration of analyte in the sample by comparison of the test response to standard responses produced by known concentrations of analyte. Again, the comparison may be performed with a spectrophotometer or by visual comparison to a color chart.
Reagent strips are a common form of test device for performing these types of analyses. These devices have a handle or support means to which is attached a carrier member or matrix such as filter paper, polymeric film, or the like, which is incorporated with the reagent/indicator components in a dry state. Contact with the liquid test sample rehydrates the test composition and initiates the assay reaction. The spectrophotometric response generated from the carrier member is then related to standards to give an indication of analyte concentration in the sample tested.
These test methods and devices are useful in a variety of fields where the quantitative or qualitative measurement of substances in liquid samples is of importance. The testing of biological fluids for medical and veterinary purposes, foods and beverages, environmental and waste waters are representative. Reagent strips are particularly well-known as useful aids in medical diagnosis, from the self-monitoring of blood glucose levels by diabetic individuals to routine urinary metabolite screening and quantitative blood chemistry analysis in physician's offices and clinical laboratories.
While the test results provided by these prior art methods and devices provide sufficient quantitation to serve as useful means of analysis, and the reagent strip configuration is particularly attractive because of its simplicity and ease of storage and use, the precision of such tests is limited by the need to make comparisons to standards. Particularly where the response is a color change and the comparison is made visually, the limited ability of the human eye to resolve small differences in color can introduce an undesirable error factor into quantitation. Furthermore, where the colors generated by the indicator reaction over the range of analyte concentrations of interest are highly saturated in hue, visual resolution for quantitation purposes can be significantly below optimal levels.
There are a number of attempts in the literature to devise quantitative test systems that would be self-indicating. By this is meant a test system that would provide a relatively unambiguous yes/no response or indication at a prescribed analyte concentration. Thus, if the programmed indicator response is observed, such as visual detection of the appearance of color, the indication is that the analyte is present at the predetermined concentration or greater. While the principle of self-indication is well-known, the prior art is devoid of a practical approach to constructing a test system that would yield sufficiently unambiguous yes/no responses to be truly self-indicating.
One very early approach to making a self-indicating test system employed an antagonist substance in the composition which would act on the indicator to prevent color formation below a predetermined level of analyte (U.S. Pat. No. 2,893,844). Indicators that are susceptible to reaction with an antagonist substance will also be affected by a variety of nonspecific environmental factors such as interfering substances in the sample. Such systems therefore are not sufficiently reliable as quantitative tests. Other early approaches used the principle of limiting the amount of indicator in the test composition (U.S. Pat. No. 3,006,735) or physically limiting the amount of analyte reaching the test composition by semipermeable membranes (U.S. Pat. No. 3,723,064).
More recently a number of different approaches have been suggested. U.S. Pat. Nos. 3,964,871; 4,042,329, and 4,059,407 reemphasize the desirability of self-indicating test devices wherein a plurality of test areas are arranged to give detectable responses to different levels of analyte. However, the reaction schemes offered for accomplishing self-indicating responses have notable shortcomings. The principal scheme proposed is based on the prior known use of indicator antagonist or titrant substances, which leads to the problem of sample interferences. The evolution of indicators has been towards compounds of greater and greater stability against environmental factors. As a result, the currently preferred indicators are essentially nontitratable by antagonist compounds as proposed in the subject references. Another approach offered is the complexation of analyte to prevent reaction with the indicator system. The systems proposed are relatively nonspecific for the analyte, some are reversible complexations, and some produce undesirable precipitates. No data is provided and the systems are quite unrefined.
Another more recent approach is described in U.S. Pat. No. 4,234,313 which proposes the use of indicators that go from colored to colorless upon reaction with analyte. This approach has the key disadvantage of requiring the use of limited amounts of indicator because the complete consumption of indicator is required for the detectable color change to occur. As a result, the indicator reaction kinetics are slow. Further, assigning a colorless result to be a positive result is the reverse of what the typical technician is used to in the laboratory.
U.S. Pat. No. 4,654,310 proposes the use of a nonresponsive reaction that is competitive with the indicator reaction to effectively reduce the rate of indicator response at varying levels of analyte concentration. This reference teaches the use of a catalyst-controlled secondary reaction to effectively remove competitive amounts of analyte from the indicator reaction. Several test areas would be provided with varying amounts of the catalyst and excess amounts of the reactants of the secondary reaction so that the ability of the indicator reaction to produce a response, e.g., color, depends on the amount of analyte present. The most significant limitation of this approach is that since the indicator and secondary reactions kinetically compete for analyte, the slope of the indicator response curve is decreased with detrimental effects on the ability to discriminate indicator responses at all levels of analyte.
Accordingly, there continues to be a need for a self-indicating test system that is resistant to interferences in the test sample, that provides a stable, essentially irreversible indicator response, and that does not sacrifice resolution.
A further limitation of reagent strip test devices is their general lack of uniformly good instrumental or visual resolution over the entire range of analyte concentrations of analytical significance. While oftentimes resolution at the lower end of the concentration range can be quite good, quantitation by instrumental or visual means suffers at the upper end of the range. A common cause of this phenomenon is the oversaturation of the indicator response, e.g., color, at high analyte concentrations.
Accordingly, there is a further need for visually interpreted or instrumentally read test systems that can be adjusted to provide optimal resolution of the indicator response within the range of analyte concentrations of interest.